| The oriental armyworm Mythimna separata,is a serious threat to the production ofcereals. Its control has largely relied on synthetic insecticides which, led to the decrease intheir effectiveness. In China, cantharidin, a natural compound of insect origin with a mode ofaction different from a conventional insecticide, is being developed as a bio-insecticide for thecontrol of lepidopteran pests. Its toxicological effects have already been studied in M.separata. However, its sublethal effects on physiological and population parameters have notyet been studied. The leaf dip bioassay results showed that cantharidin-AC (cantharidinacetone solution) had a high level of toxicity against M. separata and the96h LC50value was223μg/ml. The sublethal effects of cantharidin exposure for72h at LC10(77μg/ml) onphysiological and population parameters of M. sepatata were also investigated and data weresubjected to an age-stage two-sex life table. The sublethal effects of cantharidin indicatedreduction in survival rates of larval, pupal and adult stages. In addition, both male and femalemoths were observed with crippled wings in the cantharidin-treated cohort. The mean valuesof the finite rate of increase (λ), the intrinsic rate of increase (r) and the net reproductive rate(R0) were significantly lower in the treatment than in the control. The fecundity was alsostrongly affected by a sublethal cantharidin concentration. A sublethal concentration ofcantharidin may reduce the population growth of M. separata by decreasing its survival andreproduction and by increasing its generation time.Cantharidin, a well known natural compound produced by beetles of family Meloidaeand Oedemeridae, developed as bio-insecticide in China, was investigated for its effect ofcytochrome P450O-demethylase activity using sub-lethal concentration. Results showed thatcytochrome P450O-demethylase activity remained significantly high at12to48h aftertreatment. To study the gene expression of CYP6B7at molecular level, gene specific primerswere designed according to gene sequence of CYP6B7, whereas β-actin gene fromHelicoverpa armigera was used as internal control. Semi qRT-PCR results showed thatfluorescent intensity ratio of CYP6B7and control mRNA transcript level remained at1.01,1.23,1.68and1.62folds after12,24,36and48h, respectively after treatment by sub-lethaldose of cantharidin in artificial diet. In light of our experimental results we may conclude thatover-expression of P450s in general and CYP6B7in specific may be involved in resistancetowards cantharidin in H. armigera. Moreover, there is a potential risk of insecticide resistance against cantharidin in areas where P450s associated resistance has alreadydeveloped in lepidopteran.Previous studies revealed that cantharidin down-regulated alkaline phosphatase (ALP) atthe transcription level in Helicoverpa armigeraHub.(Lepidoptera; Noctuidae), however,nothing has been done so far to investigate the changes of ALP caused by cantharidin or otherputative inhibitors at protein level due to lack of a proper detection system. Now, thedeveloped antibody based detection system can be used for the detection of ALP and its levelin the lepidopteran pests with precision. In order to develop an antibody based detectionsystem, ALP of H. armigera (GenBank Accession No. EU729322) was cloned and expressed.The target gene H.a-ALP, having an open reading frame of1608bp, was reverse-transcribedfrom cDNA by the polymerase chain reaction. The open reading frame of the target gene wascloned into the pET-32a expression vector to obtain recombinant protein in Escherichia coliDE-3cells for the subsequent production of polyclonal antibody. New Zealand white rabbitswere used for production of anti-pET-32a-H.a-ALP. The production of antibody was alsooptimized by employing ELISA for titer determination. The produced antiserum wasprocessed and used as an antibody. Western blot results showed that the polyclonal antibodyproduced was capable of effectively binding target protein not only from H. armigera but alsofrom other lepidopterans such as Mythimna separata and Plutella xylostella. This antibodywas also used to detect levels of ALP within different instars of H. armigera. Thus, it isconcluded that this antibody-based assay is very useful for the effective detection of gene-specific expression. Furthermore, it may also be used to detect the expression levels and tissuelocalization of ALP, as well as in other physiological studies involving this enzyme.Cantharidin is a natural compound of novel structure with ideal insecticidal activity.Moreover cantharidin and its analogues have been of considerable interest as potent inhibitorsof the serine/threonine protein phosphatases. Cantharidin has a novel mode of action andshows great potential application value, however, its low yield originally extracted from thebeetles, as well as the great difficult in its chemical synthesis limit its application. Cantharidinanalogues synthesized by chemical synthesis methods possessing good bioactivity could beused in many areas substituting cantharidin. In this work, analogues of cantharidin weresynthesized by replacing the anhydride ring of5,6-dehydronorcantharidin with ethanol andchloroethanol to obtain3-(ethoxycarbonyl)-7-oxabicyclo [2.2.1]heptane-2-carboxylic acidand3-((2-chloroethoxy) carbonyl)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid, respectively.The structures of these compounds were characterized by1H NMR and13C NMR. |
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